Compositions and Methods of Treating Cardiac Fibrosis with Ifetroban

ABSTRACT

The present invention is directed to methods of treating, preventing, and/or ameliorating fibrosis syndrome, and in particular cardiac fibrosis, by administration of a therapeutically effective amount of ifetroban, or a pharmaceutically acceptable salt thereof.

FIELD OF THE INVENTION

The present invention is related to the use of thromboxane A₂ receptorantagonists (e.g., ifetroban) in the treatment and/or prevention offibrosis in mammals, e.g., humans, and pharmaceutical compositions forthe same comprising thromboxane A₂ receptor antagonists (e.g.,ifetroban) in an effective amount to treat and/or prevent thesediseases. In certain embodiments, the fibrosis is cardiac fibrosis.

BACKGROUND OF THE INVENTION

Fibrosis is the formation of excess fibrous connective tissue in anorgan or tissue in a reparative or reactive process. This can be areactive, benign, or pathological state, and physiologically acts todeposit connective tissue, which can obliterate the architecture andfunction of the underlying organ or tissue. Fibrosis can be used todescribe the pathological state of excess deposition of fibrous tissue,as well as the process of connective tissue deposition in healing. Whilethe formation of fibrous tissue is normal, and fibrous tissue is anormal constituent of organs or tissues in the body, scarring caused bya fibrotic condition may obliterate the architecture of the underlyingorgan or tissue.

For example, as fibrotic scar tissue replaces heart muscle damaged byhypertension, the heart becomes less elastic and thus less able to doits job. Similarly, pulmonary fibrosis causes the lungs to stiffen andimpairs lung function. Fibrotic growth can proliferate and invadehealthy surrounding tissue, even after the original injury heals. Inmost cases fibrosis is a reactive process, and several different factorscan apparently modulate the pathways leading to tissue fibrosis. Suchfactors include the early inflammatory responses, local increase infibroblast cell populations, modulation of the synthetic function offibroblasts, and altered regulation of the biosynthesis and degradationof collagen. Other factors include inflammation of the nearby tissue, ora generalized inflammatory state, with increased circulating mediators.

Fibrosis includes pathological conditions characterized by abnormaland/or excessive accumulation of fibrotic material (e.g., extracellularmatrix) following tissue damage. Fibroproliferative disease isresponsible for morbidity and mortality associated with vasculardiseases, such as cardiac disease, cerebral disease, and peripheralvascular disease, and with organ failure in a variety of chronicdiseases affecting the pulmonary system, renal system, eyes, cardiacsystem, hepatic system, digestive system, and skin.

To date, there are no commercially available therapies that areeffective in treating or preventing fibrotic disease, particularlycardiac fibrosis. Conventional treatment of most fibrosis-relateddisorders frequently involves corticosteroids, such as prednisone,and/or other medications that suppress the body's immune system. Thegoal of current treatment regimens is to decrease inflammation andsubsequent scarring. Responses to currently available treatments arevariable, and the toxicity and side effects associated with thesetreatments can be serious. Indeed, only a minority of patients respondto corticosteroids alone, and immune suppression medications are oftenused in combination with corticosteroids.

Right ventricular (RV) failure is the primary cause of death inpulmonary arterial hypertension (PAH), and is a source of significantmorbidity and mortality in other forms of pulmonary hypertension.Production of thromboxane and F2 isprostanes, both agonists of thethromboxane/prostainoid (TP) receptor, is increased in pathologicalstates increasing load stress, such as pulmonary arterial hypertension.The prostacyclin/thromboxane balance has been associated withcardioprotective effects under stress, probably through support of thecoronary arteries. While aspirin treatment can decrease both thromboxaneand prostaglandin production, it will suppress beneficial prostacyclinproduction and has no effect on isoprostane formation.

There are no approved therapies directed at preserving RV function.F-series and E-series isoprostanes are increased in heart failure andPAH, correlate to the severity of disease, and can signal through thethromboxane/prostanoid (TP) receptor, with effects from vasoconstrictionto fibrosis. Loss of RV function can progress despite treatmentsdecreasing pulmonary arterial pressure. RV response to chronic pressureoverload can take both adaptive and maladaptive forms, which oftendetermines clinical outcome. Adaptive ventricular hypertrophy withincreased protein synthesis sustains function, while fibrosis andcardiomyocyte hypertrophy can cause arrhythmias and contractiledysfunction, and maladaptive dilatation is associated with RV failure.Consequently, treatment strategies promoting adaptive hypertrophy in theface of chronic load stress could preserve cardiac function and improveoutcomes.

The development of cellular hypertrophy and myocardial fibrosis thatoccurs with chronic pressure overload is also associated with increasedoxidative stress and lipid peroxidation. The 15-F_(2t) isoprostane(8-isoPGF_(2α), or 8-isoF) is a common biomarker for oxidative stress,and its levels increase with ventricular dilatation and correlate withthe severity of heart failure. In addition to being a biomarker, it issuggested that 8-isoF and other isoprostanes can play a direct role incardiomyopathy. F-series and E-series isoprostanes are known to signalthrough the thromboxane/prostanoid (TP) receptor, with effects rangingfrom vasoconstriction to fibrosis. The cyclooxygenase (COX) products ofcyclic endoperoxide (PGH₂) and thromboxane A₂ (TxA₂) are also ligands ofthe TP receptor, and TP receptor activation contributes to cardiachypertrophy in models of chronic hypertension and decreases cardiacfunction in Gh-overexpressing mice. The TP receptor is found not only inplatelets and vessels but also the right ventricle, where receptordensity is increased in PAH patients.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide new methods ofpreventing and/or treating fibrosis and/or sclerosis in mammals, e.g.,humans.

It is an object of the present invention to provide a composition andmethod for preventing and/or treating and/or attenuating cardiacfibrosis in mammals, e.g., humans.

It is yet another object of the present invention to provide acomposition and method for reducing the effects of cardiac fibrosis inmammals, e.g., humans.

It has now been unexpectedly discovered that treatment of a mammal witha therapeutically effective amount of a thromboxane A₂ receptorantagonist (e.g., ifetroban) can prevent or attenuate cardiac fibrosisand associated sequalae. In certain embodiments, the administration ofthe therapeutically effective amount of a thromboxane A₂ receptorantagonist (e.g., ifetroban) can prevent or attenuate cardiomyopathy andcardiac failure in situations of pressure overload from inflammation andfibrosis towards a functional physiologic hypertrophy.

In accordance with the above objects, the present invention provides formethods of preventing, reversing, ameliorating or treating fibrosis byadministering a therapeutically effective amount of a thromboxane A₂receptor antagonist (e.g., ifetroban or a pharmaceutically acceptablesalt thereof (e.g., ifetroban sodium)) to a patient in need thereof.

In accordance with the above objects, the present invention provides formethods of preventing, reversing, ameliorating or treating cardiacfibrosis by administering a therapeutically effective amount of athromboxane A₂ receptor antagonist (e.g., ifetroban) to a patient inneed thereof.

In certain embodiments, the present invention is directed to a method oftreating and/or ameliorating a fibrotic disease or condition in apatient, in particular cardiac fibrosis, comprising administering to apatient in need thereof a therapeutically effective amount of athromboxane A₂ receptor antagonist to provide a desired plasmaconcentration of the thromboxane A₂ receptor antagonist (and/or itsactive metabolites) of about 0.1 ng/ml to about 100,000 ng/ml. Incertain embodiments, the therapeutically effective amount of athromboxane A₂ receptor antagonist to provide a desired plasmaconcentration of the thromboxane A₂ receptor antagonist of about 0.1ng/ml to about 10,000 ng/ml. In certain preferred embodiments, thethromboxane A₂ receptor antagonist is ifetroban or a pharmaceuticallyacceptable salt thereof, e.g., ifetroban sodium.

The invention is also directed to a method of providing cardioprotectiveeffects to a human patient(s) who is experiencing pulmonary arterialhypertension, via the administration of a thromboxane A₂ receptorantagonist as described herein.

The invention is further directed to a method of improving right heartadaptation to load stress in a human patient(s) via the administrationof a thromboxane A₂ receptor antagonist as described herein.

In certain embodiments, the thromboxane A₂ receptor antagonist comprisesa therapeutically effective amount of[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(Pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]-benzenepropanoicacid (Ifetroban), and pharmaceutically acceptable salts thereof.

The invention is further directed to a method of treating cardiacfibrosis in a mammal in need of treatment thereof, comprisingadministering a therapeutically effective amount of [1S-(1α,2α,3α,4α)]-2-[[3-[4-[(Pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]-benzenepropanoicacid (ifetroban), or a pharmaceutically acceptable salt thereof to themammal. In certain embodiments, the thromboxane A₂ receptor antagonistcomprises a therapeutically effective amount of[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(Pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]-benzenepropanoicacid, monosodium salt (Ifetroban Sodium). In certain preferredembodiments, the therapeutically effective amount of ifetroban reducesthe rate of formation of fibrotic tissue in the mammal. In certainpreferred embodiments, the mammal is a human patient. In certainpreferred embodiments, the therapeutically effective amount of ifetrobanslows the progression of myocardial fibrosis in the human patient and/orimproves the exercise capacity in the human patient and/or reduces RVfibrosis in the human patient, and/or reduces cardiomyocyte hypertrophyin the human patient, and/or provides an increased E/A ratio in thehuman patient, and/or increases cardiomyocyte diameter in the humanpatient, and/or improves or maintains a function selected from the groupconsisting of right ventricular ejection fraction (RVEF), leftventricular ejection fraction (LVEF), pulmonary dynamics, rightventricular systolic pressure (RVSP), left ventricular systolic function(LVSF), right ventricular diastolic function (RVDF), and leftventricular diastolic function (LVDF).

In certain preferred embodiments, the therapeutically effective amountof ifetroban is cardioprotective against pressure overload, by movingthe right heart towards adaptation rather than a maladaptive fibrosis,inflammation and cellular hypertrophy.

In certain preferred embodiments, the therapeutically effective amountof ifetroban attenuates left heart failure in the human patient.

In any of the methods described above and others described herein, theifetroban is preferably administered in an amount effective to provide aplasma concentration of the ifetroban (and/or active metabolites ofifetroban) of about 1 ng/ml to about 100,000 ng/ml or of about 1 ng/mlto about 10,000 ng/ml for ifetroban itself, and in some embodiments fromabout 1 ng/ml to about 1,000 ng/ml. In some embodiments, theafore-mentioned plasma concentration is a plasma concentration at steadystate. In some embodiments, the afore-mentioned plasma concentration isa maximum plasma concentration (Cmax). In certain preferred embodimentswhere the mammal is a human patient, the therapeutically effectiveamount is from about 100 mg to about 2000 mg per day, or from about 10mg or about 100 mg to about 1000 mg per day, and certain embodimentsmore preferably from about 100 to about 500 mg per day. The daily dosemay be administered in divided doses or in one bolus or unit dose or inmultiple dosages administered concurrently. In this regard, theifetroban may be administered orally, intranasally, rectally, vaginally,sublingually, buccally, parenterally, or transdermally.

The invention is further directed to a pharmaceutical compositioncomprising a thromboxane A₂ receptor antagonist (e.g., ifetroban or apharmaceutically acceptable salt thereof), the thromboxane A₂ receptorantagonist being in an amount effective to improve or maintain afunction selected from the group consisting of right ventricularejection fraction (RVEF), left ventricular ejection fraction (LVEF),pulmonary dynamics, right ventricular systolic pressure (RVSP), leftventricular systolic function (LVSF), right ventricular diastolicfunction (RVDF), and left ventricular diastolic function (LVDF) in amammal in need thereof. In certain preferred embodiments, the ifetrobansalt is ifetroban sodium.

In certain preferred embodiments, the pharmaceutical compositiondescribed above, the therapeutically effective amount is from about 10mg to about 1000 mg ifetroban per day. In certain preferred embodiments,the therapeutically effective amount is from about 100 to about 500 mgper day.

The present invention also relates to methods and compositions fortreating fibrosis in a subject(s) or patient(s) in need of treatmentthereof, particularly, cardiac fibrosis, the method comprisingadministering a therapeutically effective amount of a thromboxane A₂receptor antagonist to a subject(s) or patient(s) in need thereof. Inparticular, it relates to a method of treating or preventing a disorderthat results in fibrosis or sclerosis, in a subject(s) or patient(s) inneed of such treatment, comprising administering a compositioncomprising administering a therapeutically effective amount of athromboxane A₂ receptor antagonist to a patient in need thereof in anamount effective to reduce the rate of fibrosis or sclerosis. Furtherprovided is a method of preventing fibrosis or sclerosis in a subject(s)or patient(s) in need of such treatment, comprising administering acomposition comprising a thromboxane A₂ receptor antagonist in an amounteffective to reduce the formation of fibrotic or sclerotic tissue thatwould occur in the absence of such treatment.

In a certain embodiment, the fibrosis is associated with afibroproliferative disease selected from the group consisting of heartfibrosis, kidney fibrosis, liver fibrosis, lung fibrosis, and systemicsclerosis.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the above stated objects, it is believed thatadministration of a therapeutically effective amount of a thromboxane A₂receptor antagonist to a subject(s) or patient(s) in need thereof canprevent and/or treat fibrosis (fibrotic diseases or conditions, and inparticular, cardiac fibrosis).

Failure of the right ventricle to adapt to load stress is the directcause of mortality in pulmonary arterial hypertension. Thromboxaneproduction is increased in pathological states increasing load stresssuch as pulmonary arterial hypertension, and theprostacyclin/thromboxane balance has been associated withcardioprotective effects under stress, probably through support of thecoronary arteries. While aspirin treatment can decrease both thromboxaneand prostaglandin production, it also suppresses beneficial prostacyclinproduction and a more targeted approach may be necessary.

Fibrosis can occur in many tissues within the body, typically as aresult of inflammation or damage, and examples include: Pulmonaryfibrosis (lungs); Idiopathic pulmonary fibrosis (where the cause isunknown); cystic fibrosis; liver fibrosis or cirrhosis (liver); heartfibrosis, including endomyocardial fibrosis (heart), old myocardialinfarction (heart), atrial fibrosis (heart); and other fibroticconditions including but not limited to mediastinal fibrosis (softtissue of the mediastinum), myelofibrosis (bone marrow), retroperitonealfibrosis (soft tissue of the retroperitoneum), progressive massivefibrosis (lungs); a complication of coal workers' pneumoconiosis,nephrogenic systemic fibrosis (skin), Crohn's Disease (intestine),keloid (skin), scleroderma/systemic sclerosis (skin, lungs),arthrofibrosis (knee, shoulder, other joints), and some forms ofadhesive capsulitis (shoulder). Other names for various types ofpulmonary fibrosis that have been used in the past include chronicinterstitial pneumonitis, Hamman-Rich Syndrome, usual interstitialpneumonitis (UIP) and diffuse fibrosing alveolitis.

Symptoms of pulmonary fibrosis include shortness of breath, cough, anddiminished exercise tolerance. The severity of symptoms and theworsening of symptoms over time can vary and are at least partiallydependent upon the cause of the fibrosis.

Cirrhosis is extensive scarring (fibrosis) in the liver caused bylong-term damage. This damage is caused by inflammation, which is anormal response to some injuries like chronic viral infection or chronicalcoholism. The liver repairs the damaged areas by replacing them withscar tissue, in similar fashion to scar tissue developing during thehealing process when a subject sustains a cut on their body. Fibrosis inthe liver is different from the surrounding healthy liver tissue.Unfortunately, since scar tissue can't function as normal hepatocytes,too much scar tissue interferes with essential liver functions.Cirrhosis has many causes, but the most common is alcoholism and chronichepatitis. Some of the other causes of cirrhosis are obstructed bileducts in the liver and gallbladder, autoimmune hepatitis, and inheriteddiseases like Wilson's disease or hemochromatosis.

Liver fibrosis is a scarring process initiated in response to chronicliver disease (CLD) caused by continuous and repeated insults to theliver. Later stages of CLD are characterized by extensive remodeling ofthe liver architecture and chronic organ failure, regardless of theunderlying disease (e.g., cirrhosis, nonalcoholic steatohepatitis(NASH), primary sclerosing cholangitis (PSC)).

Idiopathic pulmonary fibrosis (IPF) is the main form of lung fibrosis.IPF is a debilitating and life-threatening lung disease characterized bya progressive scarring of the lungs that hinders oxygen uptake.

Systemic sclerosis is a degenerative disorder in which excessivefibrosis occurs in multiple organ systems, including the skin, bloodvessels, heart, lungs, and kidneys. Several forms of fibrotic diseasescause death in scleroderma patients, including pulmonary fibrosis,congestive heart failure, and renal fibrosis; each of which occurs inabout half of systemic sclerosis patients.

Fibrosis is also a leading cause of organ transplant rejection.

The phrase “therapeutically effective amount” refers to that amount of asubstance that produces some desired local or systemic effect at areasonable benefit/risk ratio applicable to any treatment. The effectiveamount of such substance will vary depending upon the subject anddisease condition being treated, the weight and age of the subject, theseverity of the disease condition, the manner of administration and thelike, which can readily be determined by one of ordinary skill in theart.

The term “thromboxane A2 receptor antagonist” as used herein refers to acompound that inhibits the expression or activity of a thromboxanereceptor by at least or at least about 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% in astandard bioassay or in vivo or when used in a therapeutically effectivedose. In certain embodiments, a thromboxane A2 receptor antagonistinhibits binding of thromboxane A₂ to the receptor. Thromboxane A2receptor antagonists include competitive antagonists (i.e., antagoniststhat compete with an agonist for the receptor) and non-competitiveantagonists. Thromboxane A2 receptor antagonists include antibodies tothe receptor. The antibodies may be monoclonal. They may be human orhumanized antibodies. Thromboxane A2 receptor antagonists also includethromboxane synthase inhibitors, as well as compounds that have boththromboxane A2 receptor antagonist activity and thromboxane synthaseinhibitor activity.

Thromboxane A₂ Receptor Antagonist

The discovery and development of thromboxane A₂ receptor antagonists hasbeen an objective of many pharmaceutical companies for approximately 30years (see, Dogne J-M, et al., Exp. Opin. Ther. Patents 11: 1663-1675(2001)). Certain individual compounds identified by these companies,either with or without concomitant thromboxane A₂ synthase inhibitoryactivity, include ifetroban (BMS), ridogrel (Janssen), terbogrel (BI),UK-147535 (Pfizer), GR 32191 (Glaxo), and S-18886 (Servier). Preclinicalpharmacology has established that this class of compounds has effectiveantithrombotic activity obtained by inhibition of the thromboxanepathway. These compounds also prevent vasoconstriction induced bythromboxane A₂ and other prostanoids that act on the thromboxane A₂receptor within the vascular bed, and thus may be beneficial for use inpreventing and/or treating hepatorenal syndrome and/or hepaticencephalopathy.

Suitable thromboxane A2 receptor antagonists for use in the presentinvention may include, for example, but are not limited to smallmolecules such as ifetroban (BMS;[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(pentylamino)carbonyl-1]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2yl]methyl]benzenepropanoic acid), as well as others described in U.S.Patent Application Publication No. 2009/0012115, the disclosure of whichis hereby incorporated by reference in its entirety.

Additional thromboxane A2 receptor antagonists suitable for use hereinare also described in U.S. Pat. No. 4,839,384 (Ogletree); U.S. Pat. No.5,066,480 (Ogletree, et al.); U.S. Pat. No. 5,100,889 (Misra, et al.);U.S. Pat. No. 5,312,818 (Rubin, et al.); U.S. Pat. No. 5,399,725 (Poss,et al.); and U.S. Pat. No. 6,509,348 (Ogletree), the disclosures ofwhich are hereby incorporated by reference in their entireties. Thesemay include, but are not limited to, interphenylene 7-oxabicyclo-heptylsubstituted heterocyclic amide prostaglandin analogs as disclosed inU.S. Pat. No. 5,100,889, including:

[1S-(1α, 2α, 3α,4α)]-2-[[3-[4-[[(4-cyclo-hexylbutyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]-hept-2-yl]methyl]benzenepropanoicacid (SQ 33,961), or esters or salts thereof;

[1S-(1α, 2α, 3α,4α)]-2-[[3-[4-[[[(4-chloro-phenyl)-butyl]amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]benzenepropanoicacid or esters, or salts thereof;

[1S-(1α, 2α, 3α,4α)]-3-[[3-[4-[[(4-cycloh-exylbutyl)-amino]carbonyl]-2-oxazolyl]-7-oxabicyclo]2.2.1]hept-2-yl]benzeneacetic acid, or esters or salts thereof;

[1S-(1α, 2α, 3α,4α)]-[2-[[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]phenoxy]aceticacid, or esters or salts thereof;

[1S-(1α, 2α, 3α,4α]-2-[[3-[4-[[(7,7-dime-thyloctyl)-amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-methyl]benzenepropanoicacid, or esters or salts thereof.

7-oxabicycloheptyl substituted heterocyclic amide prostaglandin analogsas disclosed in U.S. Pat. No. 5,100,889, issued Mar. 31, 1992, including[1S-[1α, 2α(Z), 3α,4α)]-6-[3-[4-[[(4-cyclohexylbutyl)amino]-carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α, 2α (Z), 3α,4α)]]-6-[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-2-thiazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α, 2α (Z), 3α,4α)]]-6-[3-[4-[[(4-cyclohexyl-butyl)methylamino]carbonyl]-2-oxazolyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α, 2α (Z), 3α,4α)]]-6-[3-[4-[(1-pyrrolidinyl)-carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α, 2α (Z), 3α,4α)]]-6-[3-[4-[(cyclohexylamino)-carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl-4-hexenoicacid or esters or salts thereof;

[1S-[1α, 2α (Z), 3α,4α)]]-6-[3-[4-[[(2-cyclohexyl-ethyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α, 2α (Z), 3α, 4α)]]-6-[3-[4-[[[2-(4-chloro-phenyl)ethyl]amino]carbonyl]-2-oxazolyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α, 2α (Z), 3α,4α)]-6-[3-[4-[[(4-chlorophenyl)-amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α, 2α (Z), 3α,4α)]]-6-[3-[4-[[[4-(4-chloro-phenyl)butyl]amino]carbonyl]-2-oxazolyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[11α, 2α (Z), 3α,4α)]]-6-[3-[4.alpha.-[[-(6-cyclohexyl-hexyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters, or salts thereof;

[1S-[1α, 2α (Z), 3α,4α)]]-6-[3-[4-[[(6-cyclohexyl-hexyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α, 2α (Z), 3α,4α]]-6-[3-[4-[(propylamino)-carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof

[1S-[1α, 2α (Z), 3α,4α)]]-6-[3-[4-[[(4-butylphenyl)-amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α, 2α (Z), 3α,4α)]]-6-[3-[4-[(2,3-dihydro-1H-indol-1-yl)carbonyl]-2-oxazolyl]-7-oxabicyclo(2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α, 2α (Z), 3α,4α)]]-6-[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-N-(phenylsulfonyl)-4-hexenamide;

[1S-[11α, 2α (Z), 3α,4α)]]-6-[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-2-oxazolyl]-N-(methylsulfonyl)-7-oxabicyclo[2-.2.1]hept-2-yl]-4-hexenamide;

[1S-[1α, 2α (Z), 3α,4α)]]-7-[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo(2.2.1]hept-2-yl]-5-heptenoic acid, or esters or salts thereof;

[1S-[1α, 2α (Z), 3α,4α)]]-6-[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-1H-imidazol-2-yl]-7-oxabicyclo-[2.2.1]hept-2-yl]-4-hexenoicacid or esters or salts thereof;

[1S-[1α, 2α, 3α, 4α)]-6-[3-[4-[[(7,7-dimethyloctyl)-amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α, 2α(E), 3α,4α)]]-6-[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid;

[1S-[1α, 2α, 3α,4α)]-3-[4-[[(4-(cyclohexylbutyl)-amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]heptane-2-hexanoicacid or esters or salts thereof,

[1S-[1α, 2α(Z), 3α,4α)]]-6-[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

7-oxabicycloheptane and 7-oxabicycloheptene compounds disclosed in U.S.Pat. No. 4,537,981 to Snitman et al, the disclosure of which is herebyincorporated by reference in its entirety, such as [1S-(1α, 2α(Z), 3α(1E, 3S*, 4R*), 4α)]]-7-[3-(3-hydroxy-4-phenyl-1-pentenyl)-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoicacid (SQ 29,548); the 7-oxabicycloheptane substituted aminoprostaglandinanalogs disclosed in U.S. Pat. No. 4,416,896 to Nakane et al, thedisclosure of which is hereby incorporated by reference in its entirety,such as [1S-[1α, 2α(Z), 3α,4α)]]-7-[3-[[2-(phenylamino)carbonyl]-hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoicacid; the 7-oxabicycloheptane substituted diamide prostaglandin analogsdisclosed in U.S. Pat. No. 4,663,336 to Nakane et al, the disclosure ofwhich is hereby incorporated by reference in its entirety, such as,[1S-[1α, 2α(Z), 3α,4α)]]-7-[3-[[[[(1-oxoheptyl)amino]-acetyl]amino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoicacid and the corresponding tetrazole, and [15-[1α, 2α(Z), 3α,4α)]]-7-[3-[[[[(4-cyclohexyl-1-oxobutyl)-amino]acetyl]amino]methyl]-7-oxabicyclo]2.2.1]hept-2-yl]-5-heptenoicacid;

7-oxabicycloheptane imidazole prostaglandin analogs as disclosed in U.S.Pat. No. 4,977,174, the disclosure of which is hereby incorporated byreference in its entirety, such as [1S-[1α, 2α(Z), 3α, 4α)]]-6-[3-[[4-(4-cyclohexyl-1-hydroxybutyl)-1H-imidazole-1-yl]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid or its methyl ester;

[1S-[1α, 2α(Z), 3α, 4α)]]-6-[3-[[4-(3-cyclohexyl-propyl)-1H-imidazol-1-yl]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid or its methyl ester;

[1S-[1α, 2α(X(Z), 3α,4α)]]-6-[3-[[4-(4-cyclohexyl-1-oxobutyl)-1H-imidazol-1-yl]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid or its methyl ester;

[1S-[1α, 2α(Z), 3α,4α]]-6-[3-(1H-imidazol-1-ylmethyl)-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid or its methyl ester; or

[1S-[1α, 2α(Z), 3α,4α)]]-6-[3-[[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-1H-imidazol-1-yl]methyl-7-oxabicyclo-[2.2.1]-hept-2-yl]-4-hexenoicacid, or its methyl ester;

The phenoxyalkyl carboxylic acids disclosed in U.S. Pat. No. 4,258,058to Witte et al, the disclosure of which is hereby incorporated byreference in its entirety, including4-[2-(benzenesulfamido)ethyl]phenoxy-acetic acid (BM 13,177-BoehringerMannheim), the sulphonamidophenyl carboxylic acids disclosed in U.S.Pat. No. 4,443,477 to Witte et al, the disclosure of which is herebyincorporated by reference in its entirety, including4-[2-(4-chlorobenzenesulfonamido)ethyl]-phenylacetic acid (BM 13,505,Boehringer Mannheim), the arylthioalkylphenyl carboxylic acids disclosedin U.S. Pat. No. 4,752,616, the disclosure of which is herebyincorporated by reference in its entirety, including4-(3-((4-chlorophenyl)sulfonyl)propyl)benzene acetic acid.

Other examples of thromboxane A₂ receptor antagonists suitable for useherein include, but are not limited to vapiprost (which is a preferredexample),(E)-5-[[[(pyridinyl)]3-(trifluoromethyl)phenyl]methylene]amino]-oxy]pentanoicacid also referred to as R68,070-Janssen Research Laboratories,3-[1-(4-chlorophenylmethyl)-5-fluoro-3-methylindol-2-yl]-2,-2-dimethylpropanoicacid [(L-655240 Merck-Frosst) Eur. J. Pharmacol. 135(2):193, Mar. 17,87],5(Z)-7-([2,4,5-cis]-4-(2-hydroxyphenyl)-2-trifl-uoromethyl-1,3-dioxan-5-yl)heptenoicacid (ICI 185282, Brit. J. Pharmacol. 90 (Proc. Suppl):228 P-Abs, March87), 5(Z)-7-[2,2-dimethyl-4-phenyl-1,3-dioxan-cis-5-yl]heptenoic acid(ICI 159995, Brit. J. Pharmacol. 86 (Proc. Suppl):808 P-Abs., December85),N,N′-bis[7-(3-chlorobenzeneamino-sulfony-1)-1,2,3,4-tetrahydro-isoquinolyl]disulfonylimide (SKF 88046, Pharmacologist 25(3):116 Abs., 117 Abs,August 83), (1. alpha.(Z)-2.beta., 5.alpha.]-(+)-7-[5-[[(1,1′-biphenyl)-4-yl]-methoxy]-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoicacid (AH 23848-Glaxo, Circulation 72(6):1208, December 85, levallorphanallyl bromide (CM 32,191 Sanofi, Life Sci. 31 (20-21):2261, Nov. 15,82),(Z,2-endo-3-oxo)-7-(3-acetyl-2-bicyclo[2.2.1]heptyl-5-hepta-3Z-enoicacid, 4-phenyl-thiosemicarbazone (EP092-Univ. Edinburgh, Brit. J.Pharmacol. 84(3):595, March 85); GR 32,191 (Vapiprost)-[1R-[1.alpha.(Z),2.beta., 3.beta.,5.alpha.]]-(+)-7-[5-([1,1′-biphenyl]-4-ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoicacid; ICI192,605-4(Z)-6-[(2,4,5-cis)2-(2-chlorophenyl)-4-(2-hydroxyphenyl)-1,3-dioxan-5-yl]hexenoicacid; BAY u 3405(ramatroban)-3-[[(4-fluorophenyl)-sulfonyl]amino]-1,2,3,4-tetrahydro-9H-c-arbazole-9-propanoicacid; or ONO 3708-7-[2.alpha.,4.alpha.-(dimethylmethano)-6.beta.-(2-cyclopentyl-2.beta.-hydroxyacetami-do)-1.alpha.-cyclohexyl]-5(Z)-heptenoicacid;(.+−.)(5Z)-7-[3-endo-((phenylsulfonyl)amino]-bicyclo[2.2.1]hept-2-exo-yl]-heptenoicacid (S-1452, Shionogi domitroban, Anboxan®);(−)6,8-difluoro-9-p-methylsulfonylben-zyl-1,2,3,4-tetrahydrocarbazol-1-yl-aceticacid (L670596, Merck) and(3-[1-(4-chlorobenzyl)-5-fluoro-3-methyl-indol-2-yl]-2,2-dimethylpropanoicacid (L655240, Merck).

The preferred thromboxane A2 receptor antagonist of the presentinvention is ifetroban or any pharmaceutically acceptable salts thereof.

In certain preferred embodiments the preferred thromboxane A2 receptorantagonist is ifetroban sodium (known chemically as[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(Pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]-benzenepropanoicacid, monosodium salt.

Methods of Treatment

In certain embodiments of the present invention there is provided amethod of preventing and/or treating and/or ameliorating fibrosis in oneor more organs or tissues in a patient or patient population byadministration of a therapeutically effective amount of a thromboxane A₂receptor antagonist to a patient(s) in need thereof.

The administration of a therapeutically effective amount of athromboxane A₂ receptor antagonist may be accomplished via anytherapeutically useful route of administration, including but notlimited to orally, intranasally, rectally, vaginally, sublingually,buccally, parenterally, or transdermally. In certain preferredembodiments, the thromboxane A₂ receptor antagonist is administeredparenterally. In certain further embodiments, the thromboxane A₂receptor antagonist is administered by intra-articular injection. Incertain further embodiments, the thromboxane A₂ receptor antagonist isadministered directly to the affected anatomic site. In anotherembodiment, the thromboxane A₂ receptor antagonist is administeredthrough the hepatic artery.

In certain preferred embodiments, the plasma concentrations ofthromboxane A₂ receptor antagonists range from about 0.1 ng/ml to about10,000 ng/ml. Preferably, the plasma concentration of thromboxane A₂receptor antagonists range from about 1 ng/ml to about 1,000 ng/ml.

When the thromboxane A₂ receptor antagonists is ifetroban, the desiredplasma concentration for treatment or prevention of fibrosis in certainembodiments should be greater than about 10 ng/mL (ifetroban free acid).Some therapeutic effects of thromboxane A₂ receptor antagonist, e.g.,ifetroban, may be seen at concentrations of greater than about 1 ng/mL.

The dose administered should be adjusted according to age, weight andcondition of the patient, as well as the route of administration, dosageform and regimen and the desired result.

In order to obtain the desired plasma concentration of thromboxane A₂receptor antagonists for the treatment or prevention of fibrosis, dailydoses of the thromboxane A₂ receptor antagonists preferably range fromabout 0.1 mg to about 5000 mg. In certain preferred embodiments, thedaily dose of thromboxane A₂ receptor antagonists for the treatment orprevention of fibrosis may range from about 1 mg to about 2000 mg; about10 mg to about 1000 mg; from about 100 mg to about 1000 mg; from about50 mg to about 500 mg; about 100 mg to about 500 mg; about 200 mg toabout 500 mg; about 300 mg to about 500 mg; or from about 400 mg toabout 500 mg per day.

In certain preferred embodiments, a daily dose of ifetroban sodium fromabout 10 mg to about 250 mg (ifetroban free acid amounts) will producetherapeutically effective plasma levels of ifetroban free acid for thetreatment or prevention of fibrosis.

Preferably, the therapeutically effective plasma concentration ofthromboxane A₂ receptor antagonists ranges from about 1 ng/ml to about1,000 ng/ml for the treatment or prevention of fibrosis.

When the thromboxane A₂ receptor antagonist is ifetroban, the desiredplasma concentration for providing an inhibitory effect ofA₂/prostaglandin endoperoxide receptor (TPr) activation, and thus areduction of cerebral microvascular activation should be greater thanabout 10 ng/mL (ifetroban free acid). Some inhibitory effects ofthromboxane A₂ receptor antagonist, e.g., ifetroban, may be seen atconcentrations of greater than about 1 ng/mL.

The dose administered must be carefully adjusted according to age,weight and condition of the patient, as well as the route ofadministration, dosage form and regimen and the desired result.

However, in order to obtain the desired plasma concentration ofthromboxane A₂ receptor antagonists, daily doses of the thromboxane A₂receptor antagonists ranging from about 0.1 mg to about 5000 mg shouldbe administered. Preferably, the daily dose of thromboxane A₂ receptorantagonists ranges from about 1 mg to about 1000 mg; about 10 mg toabout 1000 mg; about 50 mg to about 500 mg; about 100 mg to about 500mg; about 200 mg to about 500 mg; about 300 mg to about 500 mg; andabout 400 mg to about 500 mg per day.

In certain preferred embodiments, a daily dose of ifetroban sodium fromabout 10 mg to about 500 mg, preferably from about 10 mg to about 300 mg(ifetroban free acid amounts) will produce effective plasma levels ofifetroban free acid.

Pharmaceutical Compositions

The thromboxane A₂ receptor antagonists of the present invention may beadministered by any pharmaceutically effective route. For example, thethromboxane A₂ receptor antagonists may be formulated in a manner suchthat they can be administered orally, intranasally, rectally, vaginally,sublingually, buccally, parenterally, or transdermally, and, thus, beformulated accordingly.

In certain embodiments, the thromboxane A₂ receptor antagonists may beformulated in a pharmaceutically acceptable oral dosage form. Oraldosage forms may include, but are not limited to, oral solid dosageforms and oral liquid dosage forms.

Oral solid dosage forms may include, but are not limited to, tablets,capsules, caplets, powders, pellets, multiparticulates, beads, spheresand any combinations thereof. These oral solid dosage forms may beformulated as immediate release, controlled release, sustained(extended) release or modified release formulations.

The oral solid dosage forms of the present invention may also containpharmaceutically acceptable excipients such as fillers, diluents,lubricants, surfactants, glidants, binders, dispersing agents,suspending agents, disintegrants, viscosity-increasing agents,film-forming agents, granulation aid, flavoring agents, sweetener,coating agents, solubilizing agents, and combinations thereof.

Depending on the desired release profile, the oral solid dosage forms ofthe present invention may contain a suitable amount ofcontrolled-release agents, extended-release agents, modified-releaseagents.

Oral liquid dosage forms include, but are not limited to, solutions,emulsions, suspensions, and syrups. These oral liquid dosage forms maybe formulated with any pharmaceutically acceptable excipient known tothose of skill in the art for the preparation of liquid dosage forms.For example, water, glycerin, simple syrup, alcohol and combinationsthereof.

In certain embodiments of the present invention, the thromboxane A₂receptor antagonists may be formulated into a dosage form suitable forparenteral use. For example, the dosage form may be a lyophilizedpowder, a solution, suspension (e.g., depot suspension).

In other embodiments, the thromboxane A₂ receptor antagonists may beformulated into a topical dosage form such as, but not limited to, apatch, a gel, a paste, a cream, an emulsion, liniment, balm, lotion, andointment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples are not meant to be limiting and representcertain embodiments of the present invention.

Example 1

In this example, ifetroban sodium capsules are prepared with thefollowing ingredients listed in Table 1:

TABLE 1 Ingredients Percent by weight Na salt of Ifetroban 35 Mannitol50 Microcrystalline Cellulose 8 Crospovidone 3.0 Magnesium Oxide 2.0Magnesium Stearate 1.5 Colloidal Silica 0.3

The sodium salt of ifetroban, magnesium oxide, mannitol,microcrystalline cellulose, and crospovidone is mixed together for about2 to about 10 minutes employing a suitable mixer. The resulting mixtureis passed through a #12 to #40 mesh size screen. Thereafter, magnesiumstearate and colloidal silica are added and mixing is continued forabout 1 to about 3 minutes.

The resulting homogeneous mixture is then filled into capsules eachcontaining 50 mg, ifetroban sodium salt.

Example II

In this example, 1000 tablets each containing 400 mg of Ifetroban sodiumare produced from the following ingredients listed in Table 2:

TABLE 2 Ingredients Amount Na salt of Ifetroban  400 gm Corn Starch   50g Gelatin  7.5 g Microcrystalline Cellulose (Avicel)   25 g MagnesiumStearate  2.5 g

Example III

In this example. An injectable solution of ifetroban sodium is preparedfor intravenous use with the following ingredients listed in Tables 3aand 3b:

TABLE 3a Ingredients Amount Ifetroban Sodium 2500 mg Methyl Paraben 5 mgPropyl Paraben 1 mg Sodium Chloride 25,000 mg Water for injection q.s. 5liter

TABLE 3b Ingredients Amount Ifetroban Sodium 52.5 mg Sodium PhosphateDibasic Anhydrous 345 mg Sodium Phosphate Monobasic Anhydrous 1.0 gSodium Chloride 21.5 g Water for injection q.s. 5 liter

The sodium salt of ifetroban, buffers and sodium chloride are dissolvedin 3 liters of water for injection and then the volume is brought up to5 liters. The solution is filtered through a sterile filter andaseptically filled into pre-sterilized vials which are then closed withpre-sterilized rubber closures. Each vial contains a concentration of 50mg of active ingredient per 5 ml of solution.

Example IV Right Ventricular Adaptation to Load Stress Improved byThromboxane Receptor Antagonism

Failure of the right ventricle to adapt to load stress is the directcause of mortality in pulmonary arterial hypertension.Prostaglandin/thromboxane balance has been associated withcardioprotective effects under stress, probably through support of thecoronary arteries.

Methods:

FVB/N mice or mice with a GFP macrophage-specific lineage tracing marker(LysM-Cre) underwent pulmonary banding to directly increase load stress(or sham surgery for controls), and starting the following day receivedeither vehicle or the thromboxane receptor antagonist Ifetroban sodiumin their drinking water (25 mg/kg/day). After 2 weeks, noninvasivehemodynamics were obtained by transthoracic echocardiography andinvasive hemodynamics were obtained by pressure-volume catheterizationof the right heart prior to sacrifice. Hearts were assessed for fibrosisand macrophage infiltration. RNA from right ventricles were isolated andused for unbiased discovery of altered gene expression using AffymetrixMouse Gene 2.0 arrays performed on four groups (banded or sham).LysM-Cre mouse hearts were flash-frozen whole, cross-sectioned, andassessed by microscopy for fibrosis and macrophage infiltration.Differences between groups were assessed by ANOVA and t-tests.Correlations were assessed by Spearman correlation.

Results:

Banded mice receiving ifetroban showed hemodynamic markers of preservedright heart function under load stress, associated with a reduction infibrosis and reduction in recruitment of inflammatory cells. Themolecular basis for these effects, based on expression arrays, rested onreduction of pro-fibrotic pathways such as PDGF-D and FBXO32, andinduction of anti-fibrotic pathways including THBS4 and TGF-betainhibitors Asporin and LTBP2. There was also evidence of induction ofpathways related to functional hypertrophy, including potentialincreases in angiogenesis and reactivation of a developmental programassociated with cardiac myogenesis. All of these changes were specificto banded mice, and were not found in the sham surgery group thatreceived Ifetroban sodium. Since load stress has previously been shownto induce thromboxane production, this is likely because the pathwaysblocked by the drug were not active in sham surgery.

Conclusions:

Rodent models of right ventricular load stress to demonstrate thatphysiologic and molecular markers of adaptive response are improved bythe thromboxane receptor antagonist ifetroban sodium.

Example V

The purpose of this study was to determine whether ifetroban-mediatedblockade of the thromboxane-prostanoid receptor can affectcardiac/pulmonary function and gene expression in a mouse model ofpulmonary hypertension, in order to determine whether ifetroban isuseful to treat pulmonary hypertension in humans with early stages ofthe disease.

Experimental Methods: In this pulmonary artery banding (PAB) animalmodel, we used wildtype or Rosa26-rtTA2 mice on a normal diet. Mice wereanesthetized and subject to either PAB (20 mice) or sham surgery (10mice) at 4-6 months of age. Following the procedure, the mice were giveneither ifetroban water at 30 mg/kg/day or normal drinking water, andafter 14 days of treatment we performed cardiovascular phenotypingfollowed by terminal sacrifice, histology and gene expression arrays.

Echocardiograms to assess cardiac output were obtained the day prior tocatheterization and sacrifice. Our standard cardiovascular phenotypingcontinued with systemic pressure measurement by tail vein cuff, andintrajugular heart catheterization with measurement of right ventricularsystolic pressure (RVSP) for peak pressure, diastolic pressure,diastolic time constant, arterial elastance, ejection fraction, andstroke volume.

Following sacrifice, right ventricular hypertrophy was assessed in eachanimal as a percentage of heart weight. A random fraction of the righthearts were fixed in formalin for histology and examined for indices offibrosis; other hearts were snap frozen for gene expression analysis viaRNA microarray. Right hearts and lungs were collected for analysis ofprotein expression and gene expression array. Left lungs were fixed andstained. Platelet deposition was examined in small pulmonary arteryvessels. The remaining organs were harvested and frozen in liquidnitrogen for potential gene expression analysis.

The results obtained were as follows. The banding of the pulmonaryartery was successful (i.e., right ventricular systolic pressure (RVSP)increased), but ifetroban did not have an effect. Total cardiac outputwas essentially unchanged following banding, indicating right heartcompensation. The ratio of Tricuspid E to A Wave (p<0.05 by unpaired ttest) demonstrated that ifetroban significantly increased the ratio ofthe tricuspid E to A wave in banded mice. This data suggests thatifetroban increases the size of the heart in the midst of a fibroticstressor, which could be beneficial. The results also demonstrate thattreatment with Ifetroban decreases fibrosis in banded right ventricles.A cross-section of the right ventricle in the mice (trichrome-stained RVtaken at 20×) showed that ifetroban reduced fibrosis in the rightventricle for both the sham surgery and the banded right ventricle.Vehicle treated mice had increased levels of fibrotic collagen depositswhile ifetroban-treated mice showed very little fibrosis.

Genes altered by pulmonary banding and Ifetroban treatment fit intothree general categories: inflammation, fibrosis, and muscularization.In general, all three are increased by banding, but treatment withIfetroban decreases expression of pro-fibrotic and inflammatory genes,and increases expression of pro-muscularization genes.

As part of this study, RNA extracted and expression analysis wasperformed using Mouse Genone 2.0 Affymetrix expression arrays. (FIG.4C). The Array design was 2×2×2: Sham/Banded×Vehicle/Ifetroban×M/F. Intotal, there were 8 arrays; each array was a pool of RV from 3 mice.Only mice with an RVSP>30 were used for the banded groups. Bandingitself altered 199 genes (ABS (Diff in averages)—Sum of standarddeviations)>0.4 comparing vehicle banded to sham, and expression of 49of those genes was enhanced by ifetroban (difference >0.4) andexpression of 29 of those genes was attenuated by ifetroban(difference >0.4). Of the genes whose increase is blocked by ifetroban,it is assumed that about half indicate reduced inflammatory cellrecruitment, based on the fact that (i) ifetroban reduces the expressionof PDGF, a profibrotic signaling molecule (Ponten, et al.,Platelet-derived growth factor D induces cardiac fibrosis andproliferation of vascular smooth muscle cells in heart-specifictransgenic mice” Circ. Res. 2005 Nov. 11:97 (10): 1036-45); (ii) withrespect to fibrosis/anti-fibrosis, ifetroban reduces the expression ofFBX032, a profibrotic signaling molecule (Usui, et al., “Endogenousmuscle atrophy F-box mediates pressure overload-induced cardiachypertrophy through regulation of nuclear factor-kappaB”, Circ. Res.,2011 Jul. 8 09(2): 161-71); (iii) with respect tofibrosis/anti-fibrosis, ifetroban enhances the expression of certaingenes related to collagen and cell-cell adhesion, particularlythrombospondin-4 (Thbs4). Of genes whose increase was enhanced byifetroban, about half are related to collagen and cell-cell adhesion;(iv) ifetroban also enhanced the expression of genes involved with theinhibition of TGF-Beta, Asporin and Ltbp2; (v) ifetroban enhances theexpression of other genes, i.e., Nmrk2, Meox1, Nkd2, and Pkhd1; (vi)with respect to muscularization, ifetroban enhances the expression ofNMRK2, which may play a key role in controlling the progression ofmuscle differentiation (Li, et al. “A novel muscle-specific beta 1integrin binding protein (MIBP) that modulates myogenicdifferentiation), J. Cell Biol. 1999 Dec. 27: 147(7); 1391-8; (vii) withrespect to muscularization, ifetroban enhances expression of Meox1(Mankoo, et al., “The concerted action of Meox homeobox genes isrequired upstream of genetic pathways essential for the formation,patterning and differentiation of somites”, Development, 2003 October:130(19):4655-64; ifetroban enhances the expression of NKD2, Hu, et al.,“Myristoylated Naked2 antagonizes Wnt-beta-catenin activity by degradingDishevlled-1 at the plasma membrane”, J. Bio. Chem. 2010 Apr. 30:285(18) pp 0.13561-8, see FIG. 12; ifetroban enhances the expression ofPkhd1 (Gilles sen-Kaesbach, et al., “New autosomal recessive lethaldisorder with polycystic kidnesy type Potter I, characteristic face,microephaly, brachymelia, and congenital heart defects”, AM J MedGenet., 1993 Feb. 15; 45(4):511-8.

Therefore, the protective effects of ifetroban in RV stress may includereduction in recruitment of inflammatory cells; reduced induction ofpro-fibrotic pathways such as PDGF-D and FBXO32; induction ofanti-fibrotic pathways including THBS4 and TGF-beta inhibitors Asporinand LTBP2; and induction of pathways related to functional hypertrophy,including potentially angiogenesis.

Example VI

Wild-type mice underwent pulmonary artery banding followed by two weeksor six weeks of treatment with ifetroban (30 mg/kg/day via the drinkingwater) vs. control (plain drinking water). The pulmonary artery bandmimics pulmonary hypertension and right heart failure. Right hearthistology revealed significantly increased cardiomyocyte size in thecontrol mice, and ifetroban treatment was able to prevent this effectand showed similar cardiomyocyte size to animals that underwent shamsurgery.

Echocardiography after 6 weeks of treatment/pulmonary artery bandingrevealed left ventricular failure in the control mice while ifetrobantreated mice had cardiac function values similar to the animals thatunderwent sham surgery.

Right ventricular systolic pressure (RVSP) was first shown to beelevated in all of the animals which had received the pulmonary arteryband (as expected). Echo data (including end diastolic volume, endsystolic volume, left ventricular stroke volume, ejection fraction andfractional shortening) at 6 weeks revealed that the left ventricle wasfailing in the vehicle-treated PAB mice, while the ifetroban-treated PABmice were protected from these effects and had echo values similar tosham surgery animals. The fact that by extending pulmonary arterybanding out to 6 weeks there was left ventricular failure that wasn'tseen in the 2 week model is considered to be surprising, and makesifetroban's ability to protect against this left ventricular failureeven more striking. Ifetroban also protected against increases incardiomyocyte diameter in the right ventricle and reduced rightventricular fibrosis at 6 weeks.

These results suggest that ifetroban provides protection from dilatedcardiomyopathy in this 6 week model via multiple mechanisms/pathways.

Example VII

Given the predominantly deleterious consequences of TP receptoractivation in conditions of cardiac stress, and the production ofisoprostanes associated with cardiomyopathy, we examined the effects ofTP receptor antagonism in a pulmonary artery banding (PAB) model ofright ventricular pressure overload. In this Example, mice with RVdysfunction due to pressure overload by pulmonary artery banding (PAB)were given vehicle or ifetroban. Two weeks following PAB,ifetroban-treated mice were protected against pressure overload. Geneexpression arrays, quantitative histology and morphometry, lineagetracing, and cell culture systems were used to determine the mechanismof ifetroban protection. Ifetroban caused a near normalization offibrotic area, prevented cellular hypertrophy while allowing increasedRV mass, increased expression of anti-fibrotic thrombospondin-4, andblocked induction of the pro-fibrotic TGF-beta pathway. Low-dose aspirinfailed to replicate these results. Extending treatment with TP receptorantagonist to 6 weeks after PAB led to more functional adaptation anddecreased indications of cardiac failure seen with prolonged pressureoverload.

Both male and female age-matched C57Bl/6 or FVB/NJ mice, obtained byin-house breeding, were used for pulmonary artery banding. LysM-GFP micewere obtained by cross-breeding LysM-Cre×mTomato/GFP reporter miceB6.129P2-Lyz2tm1(cre)Ifo/J (a gift from TimBlackwell)×Gt(ROSA)26Sortm4(ACTB-tdTomato, -EGFP)Luo (JAX stock#007676). The day following surgery, mice were given either 25 mg/kg/dayIFETROBAN (Cumberland Pharmaceuticals Inc., Nashville, Tenn.) indrinking water or normal drinking water (vehicle) for 2 weeks or 6 weeksprior to hemodynamic evaluation. Mice were weighed and water was changedonce a week.

Results: Increased Ventricular Efficiency in PAB Mice ReceivingIfetroban

Mice underwent pulmonary arterial banding (PAB) or sham surgery, andwere treated for 2 weeks with vehicle or ifetroban (competitiveantagonist of TPα/TPβ) in the drinking water. Right ventricular systolicpressure (RVSP) was elevated in PAB mice, indicating successfulmechanical vasoconstriction. At two weeks post-banding, there was nosignificant change in cardiac output, and RV mass similarly increased invehicle- and ifetroban-treated PAB groups. However, by echocardiography,the E/A wave ratio was increased in mice given TP antagonist, suggestingincreased filling efficiency. TP receptor expression in the RV wasanalyzed by Western blot, and remained constant after 2 weeks followingPAB or drug treatment.

PAB induced an increase in RVSP from −22 mm Hg to approximately 40 mm Hg(**, p<0.01 by two-way ANOVA). Cardiac output was not reduced in PABmice after 2 weeks. The ratio of right ventricular weight to leftventricle+septum weight increases in PAB mice (**, p<0.01 by two-wayANOVA); this increase is not blocked by ifetroban. Tricuspid E/A waveratio, a measure of contractility, is improved by ifetroban (*, p<0.05by ANOVA), but only in PAB mice. TP receptor protein is expressed inwhole RV, and expression does not change with PAB.

Decreased Cardiac Fibrosis with TP Receptor Antagonism

An RVSP of 30 mmHg was used to define PAH, and only samples from mice ator above this threshold were used for analysis of PAB groups. PAB micedeveloped significant RV fibrosis by 2 weeks, which was almostcompletely abolished with ifetroban treatment. An oral dose of aspirin(10 mg/kg/day), failed to prevent fibrosis in this model suggesting thatthromboxane A2 is not the ligand mediating fibrosis. Two weeks afterPAB, fibrosis in the RV of untreated mice has increased from <5% to −20%(blue stain) in histologic sections of frozen RV stained with Masson'strichrome. This effect is blocked by ifetroban, but not by aspirin.

Changes in RV Gene Expression in Ifetroban-Treated PAB Mice

To determine the mechanism through which ifetroban reduced fibrosis andprotected RV contractility, we performed gene expression arrays on setsof pooled RV from mice with sham or PAB surgery, with and without drugtreatment. By principal components analysis strong effect of banding wasfound, and an effect of ifetroban only in mice that had undergonebanding. Principal Components Analysis shows strong separation of groupswith banding, with a strong effect of ifetroban only in mice withbanding. Ifetroban did not significantly change gene expression in micewith only sham surgeries. In PAB mice, ifetroban treatment enhancedtranscription of some genes, while the effect of banding on other geneswas blocked with TP receptor antagonism. In particular, there werechanges in gene expression associated with adhesion, collagenorganization, extracellular structure, and developmental processes.

Cardiac Monocytes in PAB Mice

By expression array, PAB mice given ifetroban had decreased RVexpression of Cd14, a marker for mature macrophages, as well as Tlr8 andother pro-inflammatory genes. TP receptors are also expressed onmonocytes, and blockade of these receptors may have immune-specificeffects. To determine whether the decrease in fibrosis seen with TPreceptor inhibition was due to decreased macrophage infiltration orproliferation in the RV, PAB on LysM-Cre mice expressing eGFP under theLysozymeM promoter was performed, where all cells that have ever hadmonocyte lineage are labeled with GFP, even if they have laterdifferentiated. While the majority of monocytes were located in the leftheart or septum, PAB appeared to increase the number of RV monocytes invehicle-treated and aspirin-treated mice. Flow cytometry of RV takenfrom wildtype mice revealed no significant changes with banding in anymacrophage population examined, with a non-significant trend towardincreased CD14/CD45 and F480/CD45-labeled cells and decreasedCD86-labeled CD45+ cells in vehicle-treated, but not ifetroban-treatedPAB mice compared to sham-operated controls. This mismatch to theLysM-Cre mice suggests that the cells lose their circulatingdifferentiation markers upon lodging in the heart. Gene expressionarrays showed ifetroban reduced expression of genes associated withinflammatory cell recruitment, but only in the context of pressureoverload. Transgenic LysM-Cre×flox-mTomato-flox eGFP mice were used tolabel cells of monocyte derivation in the RV. These mice express afluorescent red, except in cells derived from monocyte lineage (even ifthey have later differentiated), which express a fluorescent green. PABsignificantly increases numbers of green cells in vehicle and ASA mice,but not in ifetroban-treated mice (representative images from 2-3transgenic hearts shown). Flow sorting of whole RV for circulatingmarkers found a non-significant trend towards an increase in PA-bandedmice that was blocked by ifetroban.

Decreased Cellular Hypertrophy in IFETROBAN-Treated PAB Mice

Expression of genes associated with myogenic differentiation wasenhanced in PAB mice receiving TP receptor antagonist compared to bandedcontrols. Although RV weight increased similarly in vehicle-treated andifetroban-treated PAB mice, the increase in cardiomyocyte diameternormally associated with PAB was blocked with ifetroban treatment.Aspirin-treated mice had cardiomyocyte diameters similar tovehicle-treated mice. Cardiomyocyte size increases in mice with PAB.This effect is blocked by ifetroban, but not by aspirin. Western blotdemonstrated α-MHC and β-MHC expression in RV. α-MHC is variable, butunchanged, while β-MHC is strongly induced in PAB mice given the TPreceptor antagonist. Ifetroban treatment of PAB mice caused an inductionof ß-myosin heavy chain (ß-MHC) protein with no change in α-MHCexpression. In an aortic banding model of pressure overload, thisinduction is associated with decreased individual hypertrophy inß-MHC-expressing cells. The induction of ß-MHC after TP receptorantagonism was not due to increased animal age, as the average ages ofvehicle-treated and ifetroban-treated PAB mice were 688.3±19.8 and689.8±18.5 days, respectively. Gene expression arrays showedifetroban-induced genes associated with adaptive remodeling, but only inthe context of pressure overload.

Decreased TGF-ß Signaling and Enhanced TSP-4

The effect of ifetroban treatment on known fibrotic and anti-fibroticsignaling pathways was examined. Mice receiving the TP receptorantagonist had less phospho-SMAD2/3 associated with PAB thanvehicle-treated mice, and reduced expression of genes associated withfibrosis, with increased expression of antifibrotic genes.Ifetroban-treated cultured cardiomyocytes given exogenous TGF-ß had nochange in PAI-1 expression or promoter activity compared tovehicle-treated cells, indicating no direct blockade of TGF-ß signalingfrom its receptor and suggesting that the in vivo block of TGF-β occursupstream of ligand binding. Opposing the pro-fibrotic TGF-ß, pressureoverload is associated with an induction of antifibroticthrombospondin-4 (Thbs4; TSP-4) mRNA. Ifetroban treatment stronglyenhances Thbs4 expression in PAB RV only (FIG. 6B), which translates toincreased TSP-4 protein in ifetroban-treated banded mice. Whilelocalization of TSP-4 appeared to be confined to fibrotic patches invehicle-treated RV, PAB mice receiving ifetroban demonstrated increasedcardiomyocyte expression of TSP-4. Ifetroban blocks phosphorylation ofTGF-β signaling molecule Smad2/3 in vivo (each band is from a differentmouse heart). Gene expression arrays from RV showed ifetroban reducedprofibrotic genes and induced antifibrotic genes, but only in thecontext of pressure overload. In cultured cardiomyocytes, ifetroban doesnot block protein expression of canonical TGF-β target PAIL, nor does itblock induction of a luciferase activity driven by a PAH promoter. PABinduces TSP-4 expression in RV, which is strongly enhanced withifetroban treatment.

Prevention of Cardiac Failure

In the previous experiments, there was an increase in RV mass but notyet any change in cardiac output or other functional parameters, apartfrom E/A wave ratio, following PAB. To determine whether the decrease infibrosis seen with TP receptor antagonism leads to increasedpreservation of cardiac function, the period following PAB-inducedpressure overload was extended to 6 weeks and dosing with ifetroban wascontinued to 6 weeks, as well. After 6 weeks, control-treated PAB micedeveloped left ventricular dilation, as demonstrated by increasedsystolic and diastolic volume, which was accompanied by a compensatoryincreased stroke volume. This was associated with a decreased ejectionfraction and decreased fractional shortening, signs of cardiac failure.All of these were prevented in PAB mice that received ifetroban, despitesimilar RVSP. Mice underwent sham surgery or PAB and echocardiographyand pressure-loop catheterization performed after 6 weeks. End diastolicand systolic volume of the left ventricle were measured using theaverage of values calculated using both spherical and cylindricalmodels. Also obtained from echocardiography were the left ventricularstroke volume and ejection fraction, as well as percent fractionalshortening.

Comparison of High Dose Versus Low Dose Ifetroban

In another arm of this example, low dose (3 mg/kg/day) ifetroban wasadministered to mice via the drinking water for 2 weeks followingpulmonary artery banding. Right ventricular histology revealed that thislow dose treatment does not prevent fibrosis of the right ventricle. Inaddition to fibrotic collagen deposition, nuclei were present in theextracellular space from infiltrated or proliferating cells. Thepreviously presented results herein revealed an antifibrotic effect ofhigh dose ifetrobn (25 mg/kg/day) in the same model. Densitometryanalysis of thrombospondin-4 (TSP-4) Western Blots of right ventriculartissue demonstrate that high dose ifetroban (25 mg/kg/day) given via thedrinking water for 2 weeks following pulmonary artery banding increasesthe expression of antifibrotic TSP-4 while low dose ifetroban 3mg/kg/day does not.

Discussion

The effect of TP receptor antagonism was studied in mice with mechanicalconstriction of the pulmonary artery, a model of PAH-associated RVhypertrophy. Treatment with the an effective amount of the TP receptorantagonist ifetroban reduced RV fibrosis and cardiomyocyte hypertrophyin PAB mice, and increased E/A ratio, one indicator of cardiacefficiency (The E/A ratio is the ratio of the early (E) to late (A)ventricular filling velocities. In a healthy heart, the E velocity isgreater than the A velocity. In certain pathologies and with aging, theleft ventricular wall can become stiff, increasing the back pressure asit fills, which slows the early (E) filling velocity, thus lowering theE/A ratio). This was associated with augmented RV expression ofanti-fibrotic and muscularization genes, as well as decreased expressionof genes associated with inflammation and a decrease in RVphospho-SMAD2/3. Few differences were found in sham-operated micereceiving ifetroban, indicating that ifetroban-mediated gene expressionchanges are specific to PA banding. When the pressure overload wasextended to 6 weeks, mice given TP receptor antagonist were protectedfrom the indications of cardiac failure seen in vehicle-treated PABmice: gross increases in left ventricular volume as well as decreases inejection fraction and fractional shortening. This may be due toventricular interdependence, septal bowing, or neurohormonal activation.

The initial cardioprotective effects seen with TP receptor antagonismwere not duplicated with low-dose aspirin treatment of mice; suggestingthat the fibrosis associated with PAB is not mediated byplatelet-generated thromboxane. This low dose was chosen becausehigh-dose aspirin in patients with heart failure does not decreasemortality or hospitalization, and to avoid confounding anti-inflammatoryor salicylate-mediated NF-κB effects. There does remain the possibilitythat either local or macrophage production of TxA₂ is responsible forthe cardiac fibrosis observed in this model. However, isoprostanes suchas 8-iso-PGF_(2α), are also known to signal through the TP receptor, andcan cause increased collagen production and fibrogenic effects. Indeed,reducing oxidative stress and isoprostane generation through enhancementof superoxide dismutase or introduction of free radical scavengers canhave antifibrotic, cardioprotective effects in pressure-overload. Thereis some debate over which specific isoprostanes signal through the TPreceptor or whether a structurally similar “TP-like receptor” mediatessome isoprostane action. It is possible that tissue-specific receptorcomplexes may form, or there is a yet-unidentified TP-like receptor withsimilar binding that may play a role in the effects of Ifetroban.Alternatively, besides isoprostanes and thromboxane, the effects ofifetroban could be mediated by another endogenous ligand for the TPreceptor, such as prostaglandin H₂ or 20-HETE.

To further complicate matters, there are α and ß isoforms of the TPreceptor, splice variants with similar ligand binding and G_(q)/G₁₁coupling but differing localizations. Both receptors are effectivelyblocked by ifetroban. It is currently unknown which of these isoforms,and on which tissue, mediates cardiac fibrosis in response topressure-overload. By both immunoblotting and mRNA (data not shown), wedetected robust TP receptor expression in whole RV, which suggests itsexpression in cardiomyocytes, although the possible contribution offibroblast or endothelial receptors cannot be ignored. However, ourmethods were unable to distinguish between TPα and TPβ expression.

In this study, we also examined the effects of TP receptor inhibition onRV macrophage number and cell marker expression in PAB mice. By FACSanalysis we did not find any significant changes, not only withifetroban treatment compared to vehicle, but also between PAB andsham-operated mice. However, in hearts taken from mice withGFP-expressing cells of monocyte lineage, there appeared to be manymonocytes that were well-integrated with cardiomyocyte fibers, and it ispossible that we did not completely dissociate individual monocytes fromeach RV. It is also possible that the LysM-expressing cells visualizedin the transgenic hearts were actually neutrophils, although by FACS thepercentage of total CD45-expressing cells, which would includeneutrophils, did not change.

At 2 weeks post-PAB, ifetroban treatment increased the E/A wave ratio inPAB mice, indicating possible increased contractile efficiency. This issimilar to a previous study, demonstrating that TP receptor antagonismpreserves RV efficiency following endotoxic shock (Lambermont B, Kolh P,Ghuysen A, Segers P, Dogne J M, Tchana-Sato V, Morimont P, Benoit P,Gerard P, Masereel B and D'Orio V. Effect of a novel thromboxane A2inhibitor on right ventricular-arterial coupling in endotoxic shock.Shock. 2004; 21:45-51). It is unknown whether this increased efficiencywith ifetroban treatment is due to the decrease in cardiac fibrosis, orthe decrease in cardiomyocyte hypertrophy associated with PA banding, orperhaps a combination of these two mechanisms. The decreased cellularhypertrophy we find with ifetroban is consistent with the knownhypertrophic effects of TP receptor activation in mice, but is likely aconcerted in vivo effect and not due to direct TP receptor inhibition oncardiomyocytes, as a thromboxane agonist did not cause hypertrophy ofisolated cardiomyocytes in vivo.

The prevention of individual cellular hypertrophy and concomitantincreased RV expression of ß-MHC in TP antagonist-treated PAB mice, withno change in α-MHC expression, corresponds with a previous study byLopez et al (Lopez J E, Myagmar B, Swigart P M, Montgomery M D, HaynamS, Bigos M, Rodrigo MC and Simpson PC. ß-Myosin heavy chain is inducedby pressure overload in a minor subpopulation of smaller mouse cardiacmyocytes. Circ Res. 2011; 109:629-38). By size-sorting individualcardiomyocytes following pressure overload, they found that ß-MHCexpression only occurred in non-hypertrophic cardiomyocytes; all cellshad similar α-MHC expression. Although individual cells did not increasein diameter, PAB mice given ifetroban had similar increases in RV weightas vehicle-treated mice, as evidenced by increased Fulton index. Amongthe genes activated by Ifetroban in conjunction with banding were anumber of genes associated with cell growth and reprogramming, and it istheorized that TP receptor inhibition induces muscularization andfunctional hypertrophy in response to pressure overload, while alsodecreasing the fibrotic response. Decreased TGFß activity is probablypartially responsible for the decreased fibrotic response, although theifetroban-initiated event behind the decrease in TGFß is yet unknown.

TSP-4, which is increased by PA banding and further upregulated withifetroban treatment, is not known to regulate TGFß activation but itselfcan decrease fibrosis and hypertrophy, and increase contractility andcardiac adaptation in pressure overload. TSP-4 is thought to act byinducing protective endoplasmic reticulum (ER) stress signaling viaactivating transcription factor 6α (Atf6α), an ER stress responsetranscription factor. While the localization of TSP-4 to cardiomyocytesin ifetroban-treated mice, as opposed to strictly fibrotic areas invehicle-treated mice, would support intracellular signaling, in ourmodel, there was no difference in Atf6α mRNA levels either after PAbanding or TP receptor antagonism (data not shown). It is entirelypossible that the 2-week timepoint missed any increase in mRNA, or thatthe RV stress response differs from the LV model where the Atf6αresponse to TSP-4 was delineated.

In summary, these studies demonstrate that ifetroban is cardioprotectiveagainst pressure overload, by moving the right heart towards adaptationrather than a maladaptive fibrosis, inflammation and cellularhypertrophy. Protection of the right heart eventually leads toprevention of left heart failure in these mice.

In the preceding specification, the invention has been described withreference to specific exemplary embodiments and examples thereof. Itwill, however, be evident that various modifications and changes may bemade thereto without departing from the broader spirit and scope of theinvention as set forth in the claims that follow. The specification isto be regarded in an illustrative manner rather than a restrictivesense.

1. A method of treating pulmonary arterial hypertension in a human inneed of treatment thereof, comprising administering a therapeuticallyeffective amount of[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(Pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]-benzenepropanoicacid (ifetroban) or a pharmaceutically acceptable salt thereof to thehuman.
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. The method of claim 1 wherein the therapeuticallyeffective amount is from 10 mg to 1000 mg per day.
 8. The method ofclaim 7, wherein the ifetroban or the pharmaceutically acceptable saltthereof is administered orally, intranasally, rectally, vaginally,sublingually, buccally, parenterally, or transdermally. 9-19. (canceled)20. The method of claim 1, wherein the pharmaceutically acceptable saltof ifetroban is ifetroban sodium.
 21. The method of claim 7, wherein thetherapeutically effective amount is from 100 mg to 500 mg per day. 22.The method of claim 20, wherein the ifetroban sodium is in an oral soliddosage form.
 23. The method of claim 7, wherein the therapeuticallyeffective amount is 250 mg per day.
 24. The method of claim 8, whereinthe ifetroban or the pharmaceutically acceptable salt thereof isadministered orally.
 25. The method of claim 22, wherein thetherapeutically effective amount is 250 mg per day.
 26. The method ofclaim 1, wherein the pulmonary arterial hypertension is associated withsystemic sclerosis.